Antibodies to troponin i and methods of use thereof

ABSTRACT

The subject invention relates to antibodies to troponin I as well as methods of use thereof. In particular, such antibodies may be used to detect Troponin I in a patient and may also be used in the diagnosis of, for example, a myocardial infarction or acute coronary syndrome.

The present application is filed as a continuation of U.S. patentapplication Ser. No. 14/035,420, which was filed Sep. 24, 2013, which isa reissue of U.S. patent application Ser. No. 12/391,937 (now U.S. Pat.No. 8,030,026), which was filed on Feb. 24, 2009. The entire text of theaforementioned applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The subject invention relates to antibodies to troponin I as well asmethods of use thereof.

BACKGROUND INFORMATION

Troponin I is a muscle protein which may be used in the determination ofmyocardial damage subsequent to or during, for example, a myocardialinfarction. In particular, troponin I is one of three subunits of thetroponin complex which is located on the thin filament of the musclecontractile apparatus. This complex has a primary role in controllingthe process of muscle contraction. The other two subunits (i.e., T andC) are also immobilized on the thin myofilaments with troponin I incardiac as well as skeletal muscle tissue.

Assays have been described which measure cardiac troponin I in humanserum. For example, a radioassay has been used for this purpose (Cumminset al., Am Heart Journal 113:1333-1344 (1987). However, the assayutilized polyclonal antibodies having significant cross-reactivity toskeletal forms of troponin I. Further, a sandwich assay has beenutilized which uses two different monoclonal antibodies (Bodar et al.,Clinical Chemistry 38:2203-2214 (1992); see also U.S. Pat. No.7,285,418). Unfortunately, such assays have a very high degree ofimprecision. Thus, the need certainly exists for immunoassays that arehighly specific for and sensitive to troponin I. These immunoassays mustalso utilize antibodies which do not possess cross-reactivity totroponin I found in skeletal tissue. In particular, such immunoassaysare needed so that appropriate therapy can be utilized by the treatingphysician thereby giving the affected patient the best possibleprognosis.

All patents and publications referred to herein are hereby incorporatedin their entirety by reference.

SUMMARY OF THE INVENTION

The present invention pertains to binding proteins, particularlyantibodies, capable of binding to cardiac troponin I. In particular,these antibodies bind to one or more epitopes of troponin I. Further,the present invention also provides methods of producing and using thesebinding proteins or portions thereof, for example, in diagnostic assays.

In particular, the present invention encompasses a Chinese Hamster Ovary(CHO) cell line, referred to as TnI 19C7 AM1 hG1 CHO 204, designated byAmerican Type Culture Collection (ATCC) deposit number PTA-9816 as wellas the recombinant antibody produced by this cell line.

Additionally, the present invention includes an isolated binding proteincomprising an antigen-binding domain which binds to Troponin I, saidantigen-binding domain comprising at least one complementaritydetermining region (CDR) comprising an amino acid sequence selected fromthe group consisting of: GYTFTDYNLH (SEQ ID NO:52), YIYPYNGITGYNQKFKS(SEQ ID NO:53), DAYDYDLTD (SEQ ID NO:54), RTSKNVGTNIH (SEQ ID NO: 55),YASERLP (SEQ ID NO:56) and QQSNNWPYT (SEQ ID NO:57). The binding proteinof the present invention may include, for example, at least 3 of theseCDRs. Further, this binding protein may also comprise a human acceptorframework or scaffold. This binding protein may be selected from thegroup consisting of, for example, an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, a scFv, a single domain antibody, a diabody, a multispecificantibody, a dual specific antibody, an anti-idiotypic antibody, abispecific antibody, or a functionally active epitope-binding fragmentof any one of these entities.

The present invention also encompasses an isolated nucleic acid moleculeencoding a binding protein, wherein the amino acid sequence of thevariable heavy chain of the binding protein has at least 70% identity toSEQ ID NO.: 25 (see FIG. 12). This molecule may also comprise a variablelight chain having at least 70% identity to SEQ ID NO.: 28 (see FIG.12).

Furthermore, the present invention includes an isolated nucleic acidmolecule encoding a binding protein, wherein the amino acid sequence ofthe variable heavy chain of said binding protein is SEQ ID NO.:25.

Additionally, the present invention includes an isolated nucleic acidmolecule encoding a binding protein, wherein the amino acid sequence ofthe variable light chain of said binding protein is SEQ ID NO.: 28. Themolecule may further comprise an isolated nucleic acid molecule encodinga variable heavy chain, wherein the amino acid sequence of the heavychain is SEQ ID NO.: 25.

The present invention also includes a vector comprising one or more ofthe nucleic acid molecules described above, attached to a regulatoryelement (e.g., a promoter) as well as a host cell comprising thisvector.

Moreover, the present invention includes a method of producing any ofthe binding proteins described above, capable of binding to Troponin I,which method comprises culturing the host cell, described above, for atime and under conditions sufficient to produce the binding protein ofinterest. The invention also includes the binding protein produced bythis method.

Furthermore, the present invention encompasses a pharmaceuticalcomposition comprising any one or more of the binding proteins describedabove and a pharmaceutically acceptable carrier.

Also, the present invention includes a method of detecting Troponin Iantigen in a test sample. This method comprises the steps of: contactingthe test sample with an antibody which binds to Troponin I and comprisesSEQ ID NO:25 for a time and under conditions sufficient for theformation of antibody/antigen complexes; and detecting presence of thecomplexes, presence of the complexes indicating presence of Troponin Iantigen in said test sample. The antibody may further comprise SEQ IDNO:28. The antibody may be produced by a Chinese Hamster Ovary cell linehaving ATCC deposit designation PTA-9816.

The present invention also includes a method of detecting Troponin Iantigen in a test sample comprising the steps of: contacting the testsample with a first antibody which binds to Troponin I and comprises SEQID NO:25 for a time and under conditions sufficient for the formation offirst antibody/antigen complexes; adding a conjugate to the firstantibody/antigen complexes, wherein said conjugate comprises a secondantibody attached to a signal generating compound capable of generatinga detectable signal, for a time and under conditions sufficient to formfirst antibody/antigen/second antibody complexes; and detecting presenceof a signal generating by the signal generating compound, presence ofthe signal indicating presence of Troponin I antigen in said testsample. The first antibody may further comprise SEQ ID NO:28 and may beproduced by a Chinese Hamster Ovary cell line having ATCC depositdesignation PTA-9816.

Also, the present invention includes a method of detecting Troponin Iantigen in a test sample comprising the steps of: contacting Troponin Iantigen with an antibody to Troponin I for a time and under conditionssufficient to form Troponin I antigen/antibody complexes, wherein theantibody comprises SEQ ID NO:25 and is labeled with a signal-generatingcompound capable of generating a detectable signal; adding the testsample to said Troponin I antigen/antibody complexes for a time andunder conditions sufficient to form Troponin I antigen/antibody/TroponinI test sample antigen complexes; and detecting presence of a signalgenerating by the signal generating compound, presence of the signalindicating presence of Troponin I antigen in the test sample. Again, theantibody may further comprise SEQ ID NO:28 and may be produced by aChinese Hamster Ovary cell line having ATCC deposit designationPTA-9816.

The present invention also encompasses another method of detectingTroponin I antigen in a test sample. This method comprises the steps of:contacting the test sample with 1) a Troponin I reference antigen,wherein the antigen is attached to a signal generating compound capableof generating a detectable signal and 2) an antibody to Troponin Iantigen wherein the antibody comprises SEQ ID NO:25, for a time andunder conditions sufficient to form Troponin I referenceantigen/antibody complexes; and detecting a signal generated by thesignal generating compound, wherein the amount of Troponin I antigendetected in the test sample is inversely proportional to the amount ofTroponin I reference antigen bound to the antibody. Again, the antibodymay further comprise SEQ ID NO:28 and may be produced by a ChineseHamster Ovary cell line having ATCC deposit designation PTA-9816.

In addition, the present invention includes pharmaceutical compositioncomprising any one or more of the binding proteins described above and apharmaceutically acceptable carrier.

The present invention also encompasses a method of diagnosing acutecoronary syndrome or myocardial infarction in a patient suspected ofhaving one of these conditions. This method comprises the steps of:isolating a biological sample from the patient; contacting thebiological sample with an antibody which binds to Troponin I andcomprises SEQ ID NO:25, for a time and under conditions sufficient forformation of Troponin I antigen/antibody complexes; detecting presenceof the Troponin I antigen/antibody complexes; dissociating the TroponinI antigen present in the complexes from the antibody present in saidcomplexes; and measuring the amount of dissociated Troponin I antigen,wherein an amount of Troponin I antigen greater than approximately 1-5times the Troponin I value of the 99^(th) percentile of a normalpopulation indicates a diagnosis of acute coronary syndrome ormyocardial infarction in the patient.

The present invention includes an additional method of diagnosing acutecoronary syndrome or myocardial infarction in a patient suspected ofhaving one of these conditions. This method comprises the steps of:isolating a biological sample from the patient; contacting thebiological sample with a first antibody which binds to Troponin I andcomprises SEQ ID NO:25, for a time and under conditions sufficient forthe formation of Troponin I antigen/antibody complexes; adding aconjugate to the resulting Troponin I antigen/antibody complexes for atime and under conditions sufficient to allow the conjugate to bind tothe bound Troponin I antigen, wherein the conjugate comprises a secondantibody attached to a signal generating compound capable of generatinga detectable signal; detecting the presence of Troponin I antigen whichmay be present in said biological sample by detecting a signal generatedby said signal generating compound; and measuring the amount of TroponinI antigen present in the test sample by measuring the intensity of thesignal, an amount of Troponin I antigen greater than approximately 1-5times the value of the 99^(th) percentile of a normal populationindicating a diagnosis of acute coronary syndrome or myocardialinfarction in the patient.

The present invention also includes a kit comprising any one or more ofthe monoclonal antibodies or binding proteins described above and, ifneeded, instructions describing the manner in which to use this kit.

Additionally, the present invention includes an isolated binding proteinwhich comprises an antigen-binding domain, wherein the antigen-bindingdomain comprises at least one CDR comprising an amino acid sequenceselected from the group consisting of:

CDR-VH1.  (SEQ ID NO: 63) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,wherein:

X₁ is G;

X₂ is Y;

X₃ is Tor S;

X₄ is F;

X₅ is T;

X₆ is D;

X₇ is Y;

X₈ is N;

X₉ is I or L; and

X₁₀ is H.

CDR-VH2. (SEQ ID NO: 64) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇,wherein:

X₁ is Y;

X₂ is I;

X₃ is Y;

X₄ is P;

X₅ is Y;

X₆ is N;

X₇ is G;

X₈ is I;

X₉ is T;

X₁₀ is G;

X₁₁ is Y;

X₁₂ is N;

X₁₃ is Q;

X₁₄ is K;

X₁₅ is F;

X₁₆ is K; and

X₁₇ is S.

CDR-VH3. (SEQ ID NO: 65) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,wherein:

X₁ is D;

X₂ is A or F;

X₃ is Y;

X₄ is D;

X₅ is Y or S;

X₆ is D;

X₇ is W, Y or A;

X₈ is L;

X₉ is A or T; and

X₁₀ is Y or D.

CDR-VL1. (SEQ ID NO: 66) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,wherein:

X₁ is R;

X₂ is A or T;

X₃ is S;

X₄ is Q or K;

X₅ is S or N;

X₆ is I or V;

X₇ is G;

X₈ is T;

X₉ is N;

X₁₀ is I; and

X₁₁ is Y or H.

CDR-VL2. (SEQ ID NO: 67) X₁-X₂-X₃-X₄-X₅-X₆-X₇,wherein:

X₁ is Y;

X₂ is A or G;

X₃ is S or T;

X₄ is E;

X₅ is S or R;

X₆ is I, L or V; and

X₇ is S, P or F.

and

CDR-VL3. (SEQ ID NO: 68) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉,wherein:

X₁ is Q;

X₂ is Q;

X₃ is S;

X₄ is N;

X₅ is N;

X₆ is W;

X₇ is P;

X₈ is Y; and

X₉ is T.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart showing the steps used to identify and createantibodies that have improved affinity for troponin I.

FIGS. 2A and 2B are the nucleotide description (SEQ ID NO:109 and SEQ IDNO:110) of the wild-type TnI 19C7 single-chain variable fragment(“scFv”).

FIG. 3 shows that yeast expressing full-length TnI 19C7 single-chainvariable fragment (scFv) bind to single chain troponin I(28-110aa)-linker-troponin C known as scTnI-C-2 (Spectral diagnostics,RP-3700). More specifically, this figure shows that TnI 19C7 scFvexpressing yeast were incubated with scTnI-C-2 or anti-V5, followed byeither anti-troponin mAb and goat anti mouse-phycoerythrin (GAM:PE)(FIG. 3B) or GAM:PE respectively (FIG. 3A). The flow cytometryhistograms illustrate the full-length expression of TnI 19C7 scFv asdetected by anti-V5 and the ability of TnI 19C7 scFv to bind toscTnI-C-2. PE-A units (abscissa): 10², 10³, 10⁴, and 10⁵. Count units(ordinate): 10², 10³, 10⁴, and 10⁵.

FIG. 4 shows the TnI 19C7 scFv off-rate measurement. More specifically,yeast expressing TnI 19C7 scFv were incubated with a saturatingconcentration of scTnI-C-2. Cells were washed twice and at each timepoint, cells were transferred to ice, washed and incubated with anti-TnImAb. After 30 minutes, cells were washed again and incubated with goatanti-mouse phycoerythrin. Again after 30 minutes, cells were washed andanalyzed on the flow cytometer. A first order decay equation was used tofit the individual time points where m1 was the theoretical maximum meanfluorescence units (“MFU”) at time 0, m2 was the off-rate (“koff”), m3was the background MFU due to autofluorescence and M0, which is the timex (the x being the time that is being measured) was the time x thatmeasurements are taken. The half-life (t_(1/2)) of TnI 19C7 scFv bindingto TnI-C-2 was calculated using: t_(1/2)=1n2/k_(off). Five times thehalf-life was the time used to sort the TnI 19C7 scFv CDR mutageniclibraries.

FIG. 5 shows the TnI 19C7 scFv equilibrium dissociation constant (KD)measurement. More specifically, yeast expressing TnI 19C7 scFv wereincubated with varying concentrations of scTnI-C-2. Cells were washedtwice with PBS pH6.8/2% BSA/0.02% Standapol ES-1 and incubated withanti-TnI mAb for 30 min. Cells were washed again and incubated with goatanti-mouse phycoerythrin for 30 min. Finally, cells were washed andanalyzed on the flow cytometer.

FIG. 6 is a schematic depiction that shows how degenerateoligonucleotides were designed so that primers are made such that foreach CDR nucleotide residue 70% remains the wild-type residue and 30% amix of the other three residues. Two PCR products are generated for eachlibrary a spiked (sp) PCR product and a non-spiked PCR product. Thespiked and non-spiked PCR products are combined to generate an intactCDR mutagenized scFv library.

FIG. 7 is a schematic depiction that shows how the TnI 19C7 scFv librarywas constructed using yeast homologous recombination. More specifically,the spiked CDR PCR product and the excised yeast display vector weretransformed into S. cerevisiae strain EBY100. Transformed clones wereselected in tryptophan deficient glucose media.

FIGS. 8A and 8B are a summary showing the PCR primers that were used togenerate the scFv construct (tpVHfor through tpVLrev), those used togenerate the CDR spiked libraries (19H1spfor through pYD41rev2) andthose used to generate the combination library (19FRH2for to 19FRL3)(see SEQ ID Nos:1-22). The bold and enlarged areas of the primersrepresent those regions in which a “70% wild-type, 30% other nucleotidemixture” was incorporated while the primers were being made. Such a“spiked” primer generated the diversity within the library.

FIG. 9 shows equilibrium dissociation constant (KD) measurements ofselected TnI 19C7 scFv determined as described above in FIG. 5.

FIG. 10 shows the results of relative antibody affinity as measured asan antigen 50% (Ag50). Four TnI 19C7 clones were converted into mouseIgG2ak antibodies by cloning the variable domains onto theimmunoglobulin constant domains. Antibodies were expressed in atransient HEK 294 cell system. The Ag50 is the concentration of scTnI-Cat which is 50% of the maximum signal and represents the relativeaffinity ranking of the selected TnI 19C7 AM candidates. TnI 19C7 AM1exemplifies the tightest relative affinity compared to the TnI 19C7wild-type antibody.

FIG. 11 illustrates TnI 19C7 AMI's ability to bind to scTnI-C in anARCHITECT® assay format (Abbott Laboratories, Abbott Park, Ill.). TnI19C7 was labeled with acridinium and assayed for binding to scTnI-Cusing anti-TnI capture beads. (X=signal generated with given calibratorconcentration of scTnI-C; X/A=ratio of calibrator X signal to calibratorA signal; RLU=Relative Light Units). TnI 19C7 AM1 exhibited betterbinding in this assay format for the range of calibrators compared tothe wild-type TnI 19C7 antibody.

FIGS. 12A and 12B illustrate the nucleotide (SEQ ID NO:23, SEQ ID NO:24(complement), SEQ ID NO:26 and SEQ ID NO:27 (complement)) and encodedamino acid sequences of the heavy (SEQ ID NO:25) and light (SEQ IDNO:28) chains of monoclonal antibody TnI 19C7 AM1 and, in particular, ofthe complementarity determining regions (CDRs).

FIG. 13 illustrates the positions within the heavy and light chains ofthe TnI 19C7 CDRs that may be substituted with amino acids other thanthose shown in FIG. 12 (SEQ ID Nos: 30-49).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear; however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

In order that the present invention may be more readily understood,select terms are defined below.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The subject invention also includes isolated nucleotide sequences (orfragments thereof) encoding the variable light and heavy chains of theantibodies described herein as well as those nucleotide sequences (orfragments thereof) having sequences comprising, corresponding to,identical to, hybridizable to, or complementary to, at least about 70%(e.g., 70% 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%), preferably atleast about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or89%), and more preferably at least about 90% (e.g, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100%) identity to these encoding nucleotidesequences. (All integers (and portions thereof) between and including70% and 100% are considered to be within the scope of the presentinvention with respect to percent identity.) Such sequences may bederived from any source (e.g., either isolated from a natural source,produced via a semi-synthetic route, or synthesized de novo). Inparticular, such sequences may be isolated or derived from sources otherthan described in the examples (e.g., bacteria, fungus, algae, mouse orhuman).

In addition to the nucleotide sequences described above, the presentinvention also includes amino acid sequences of the variable light andheavy chains of the antibodies described herein (or fragments of theseamino acid sequences). Further, the present invention also includesamino acid sequences (or fragments thereof) comprising, correspondingto, identical to, or complementary to at least about 70% (e.g., 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%), preferably at leastabout 80% (e.g., 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%), andmore preferably at least about 90% identity (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%), to the amino acid sequences ofthe proteins of the present invention. (Again, all integers (andportions thereof) between and including 70% and 100% (as recited inconnection with the nucleotide sequence identities noted above) are alsoconsidered to be within the scope of the present invention with respectto percent identity.)

For purposes of the present invention, a “fragment” of a nucleotidesequence is defined as a contiguous sequence of approximately at least6, preferably at least about 8, more preferably at least about 10nucleotides, and even more preferably at least about 15 nucleotidescorresponding to a region of the specified nucleotide sequence.

The term “identity” refers to the relatedness of two sequences on anucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments (or two amino acid sequences).“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math. 2:482 (1981), by the algorithm of Needleman &Winch, J. Mol. Biol. 48:443 (1970), by the method of Pearson & Lipmann,Proc. Natl. Acad. Sci. (USA) 85:2444 (1988) and by computer programswhich implement the relevant algorithms (e.g., Crustal Macaw Pileup(Higgins et al., CABIOS. 5LI51-153 (1989)), FASTDB (Intelligentsias),BLAST (National Center for Biomedical Information; Latches et al.,Nucleic Acids Research 25:3389-3402 (1997)), PILEUP (Genetics ComputerGroup, Madison, Wis.) or GAP, BESTFIT, FASTA and TFASTA (WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, Madison,Wis.). (See U.S. Pat. No. 5,912,120.)

For purposes of the present invention, “complementarity” is defined asthe degree of relatedness between two DNA segments. It is determined bymeasuring the ability of the sense strand of one DNA segment tohybridize with the anti-sense strand of the other DNA segment, underappropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

In the double helix, adenine appears in one strand, thymine appears inthe other strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of the two DNA segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence, or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

“Biological activity” as used herein, refers to all inherent biologicalproperties of an antibody against troponin I or troponin I. Suchproperties include, for example, the ability of the antibody to bind totroponin I and functionally-related antibodies described herein.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody entities are known in the art, non-limitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved; termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass and may be from any species (e.g., mouse, human,chicken, rat, rabbit, sheep, shark and camelid).

The CDRs of the antibodies of the present invention are shown in Tables1 and 2 below:

TABLE 1 CDRs OF HEAVY CHAIN FOR TnI 19C7 AM1 SEQ ID No. Protein regionSequence 52 CDR H1 GYTFTDYNLH 53 CDR H2 YIYPYNGITGYNQKFKS 54 CDR H3DAYDYDYLTD

TABLE 2 CDRs OF LIGHT CHAIN FOR TnI 19C7 AM1 SEQ ID No. Protein regionSequence 55 CDR L1 RTSKNVGTNIH 56 CDR L2 YASERLP 57 CDR L3 QQSNNWPYT

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by one or more fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific, specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., Intern. Appln.Public. No. WO 90/05144 A1 herein incorporated by reference), whichcomprises a single variable domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed herein within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodies,are also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art, and examples are presented in Table 3.

TABLE 3 SEQUENCE OF HUMAN IgG HEAVY CHAIN CONSTANT DOMAIN ANDLIGHT CHAIN CONSTANT DOMAIN Sequence Sequence123456789012345678901234567 Protein Identifier 89012 Ig gamma-1SEQ ID NO.: 50 ASTKGPSVFFLAPSSKSTSGGTAALGC constant LVKDYFPEPVTVSWNSGALTSGVHTFP region AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL Sequence Sequence Protein Identifier SLSPGKIg gamma-1 SEQ ID NO.: 51 ASTKGPSVFPLAPSSKSTSGGTAALGC constantLVKDYFPEPVTVSWNSGALTSGVHTFP region AVLQSSGLYSLSSVVTVPSSSLGTQTY mutantICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK Ig Kapa SEQ ID NO.: 61TVAAPSVFIFPPSDEQLKSGTASVVCL constant LNNFYPREAKVQWKVDNALQSGNSQES regionVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Ig LambdaSEQ ID NO.: 62 QPKAAPSVTLFPPSSEELQANKATLVC constantLISDFYPGAVTVAWKADSSPVKAGVET region TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds troponin I is substantially free of antibodies that specificallybind antigens other than troponin I). An isolated antibody thatspecifically binds troponin I may, however, have cross-reactivity toother antigens, such as troponin I molecules from other species.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedbelow), antibodies isolated from a recombinant, combinatorial humanantibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; AzzazyH., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H.,and Chames P. (2000) Immunology Today 21:371-378), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. AcidsRes. 20: 6287-6295; Kellermann, S-A. and Green, L. L. (2002) CurrentOpinion in Biotechnology 13:593-597; Little M. et al (2000) ImmunologyToday 21:364-370) or antibodies prepared, expressed, created or isolatedby any other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species. The present invention encompasseschimeric antibodies having, for example, murine heavy and light chainvariable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3. (In addition, for purposes of the presentinvention, the AbM definition as defined by Oxford Molecular's ABMantibody modeling software was used to define the CDR-H1 region fromamino acids 26-35 for the heavy chain.)

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, such as AbM definitions, butwill nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat, AbM or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (.J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

In one embodiment of the invention, the murine heavy chain and lightchain donor sequences are selected from the sequences described below:

TABLE 4 HEAVY CHAIN DONOR SEQUENCES FOR TnI 19C7 AM1 Sequence SEQ ID NO.12345678901234567890123456789012 69 EVTLRESGPALVKPTQTLTLTCTFSGFSLS 70WIRQPPGKALEWLA 71 RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR 72 WGQGTTVTVSS 73EVTLKESGPVLVKPTETLTLTCTVSGFSLS 74 WIRQPPGKALEWLA 75RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR 76 WGQGTTVTVSS 77EVQLVESGGGLVQPGGSLRLSCAASGFTFS 78 WVRQAPGKGLEWVG 79RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR 80 WGQGTTVTVSS 81EVQLVESGGGLVKPGGSLRLSCAASGFTFS 82 WVRQAPGKGLEWVS 83RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 84 WGQGTTVTVSS 85EVQLVQSGAEVKKPGSSVKVSCKASGGTFS 86 WVRQAPGQGLEWMG 87RVTITADKSTSTAYMELSSLRSEDTAVYYCAR 88 WGQGTTVTVSS 89EVQLVQSGAEVKKPGASVKVSCKASGYTFT 90 WVRQAPGQGLEWMG 91RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR 92 WGQGTTVTVSS

TABLE 5 LIGHT CHAIN DONOR SEQUENCBS FOR TnI 19C7 AM1 Sequence SEQ ID NO.12345678901234567890123456789012  93 DIVMTQSPDSLAVSLGERATINC  94WYQQKPGQPPKLLIY  95 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC  96 FGGGTKVEIKR  97EIVMTQSPATLSVSPGERATLSC  98 WYQQKPGQAPRLLIY  99GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 100 FGGGTKVEIKR 101DIQMTQSPSSLSASVGDRVTITC 102 WYQQKPEKAPKSLIY 103GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 104 FGGGTKVEIKR 105DIQMTQSPSSVSASVGDRVTITC 106 WYQQKPGKAPKLLIY 107GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 108 FGGGTKVEIKR

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med BioL. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the recognition that germline antibody genes aremore likely than mature antibody genes to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as from a foreign source when usedtherapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, preferably at least 85%, more preferably atleast 90%, more preferably at least 95%, more preferably at least 98%and most preferably at least 99% identical to the amino acid sequence ofa non-human antibody CDR. A humanized antibody comprises substantiallyall of at least one, and typically two, variable domains (Fab, Fab′,F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. Preferably, a humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. In someembodiments, a humanized antibody contains both the light chain as wellas at least the variable domain of a heavy chain. The antibody also mayinclude the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Inother embodiments, a humanized antibody only contains a humanized lightchain. In some embodiments, a humanized antibody only contains ahumanized heavy chain. In specific embodiments, a humanized antibodyonly contains a humanized variable domain of a light chain and/orhumanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-troponin I antibody that binds to troponin I.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl and, in certain embodiments, may have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(d)” or “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. Preferably, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g. ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. The terms“crystal”, and “crystallized” as used herein, refer to an antibody, orantigen-binding portion thereof, that exists in the form of a crystal.Crystals are one form of the solid state of matter, which is distinctfrom other forms such as the amorphous solid state or the liquidcrystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well-understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nded., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double-stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences that are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells that transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell but also to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae or Picchia pastoris.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of troponin I).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of troponin I). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in International ApplicationPublication No. WO 01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, troponin I polypeptides, nucleic acids, carbohydrates, orany other molecules that bind to troponin 0.1.

The term “antagonist” or “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other mammalian or non-mammaliananimals. Such substances include, but are not limited to, blood, serum,urine, synovial fluid, cells, organs, tissues (e.g., brain), bonemarrow, lymph nodes, cerebrospinal fluid, and spleen.

Methods of Making Antibodies

Antibodies of the present invention may be made by any of a number oftechniques known in the art. For example, antibodies can be preparedusing a wide variety of techniques including the use of recombinant orphage display technologies, or a combination thereof. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

In one embodiment, the present invention provides a method of generatingrecombinant antibodies (as well as antibodies produced by the method)comprising culturing a Chinese Hamster Ovary cell line secreting anantibody of the invention.

Further, fragments of the antibody of the present invention whichrecognize specific epitopes may be generated by known techniques. Forexample, Fab and F(ab′)2 fragments of the invention may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the, CHI domain of the heavy chain.

Production of Anti-Troponin I Antibodies Using Recombinant AntibodyLibraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, Ladner et al., U.S. Pat.No. 5,223,409; Kang et al., International Appln. Publication No. WO92/18619; Dower et al., International Appln. Publication No. WO91/17271; Winter et al., International Appln. Publication No. WO92/20791; Markland et al., International Appln. Publication No. WO92/15679; Breitling et al., International Appln. Publication No. WO93/01288; McCafferty et al., PCT Publication No. WO 92/01047; Garrard etal., International Appln. Publication No. WO 92/09690; Fuchs et al.(1991), Bio/Technology 9:1370-1372; Hay et al., (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989), Science 246:1275-1281;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993)EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896;Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991), Nuc Acid Res 19:4133-4137; and Barbas et al.(1991), PNAS 88:7978-7982, U.S. Patent Application Publication No.20030186374, and International Application Publication No. WO 97/29131,the contents of each of which are incorporated herein by reference.

The recombinant antibody library may be from a subject immunized withtroponin I, or a portion thereof. Alternatively, the recombinantantibody library may be from a naive subject, i.e., one who has not beenimmunized with troponin I, such as a human antibody library from a humansubject who has not been immunized with human troponin I. Antibodies ofthe invention are selected by screening the recombinant antibody librarywith the peptide comprising human troponin I to thereby select thoseantibodies that recognize troponin I. Methods for conducting suchscreening and selection are well known in the art, such as described inthe references in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for troponin I, such asthose that dissociate from human troponin I with a particular k_(off)rate constant, the art-known method of surface plasmon resonance can beused to select antibodies having the desired k_(off) rate constant.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human troponin I. In variousembodiments, the antibody is a recombinant antibody.

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed Wittrup etal., U.S. Pat. No. 6,699,658 incorporated herein by reference.

Production of Recombinant Antibodies

As noted above, antibodies of the present invention may be produced byany number of techniques known in the art. For example, expression fromhost cells, wherein expression vector(s) encoding the heavy and lightchains is (are) transfected into a host cell by standard techniques isthe preferred method of producing the antibodies of the presentinvention. (The various forms of the term “transfection” are intended toencompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like.) Although it is possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells, expression of antibodies in eukaryotic cells ispreferable, and most preferable in mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

Anti-Troponin Antibodies

The isolated anti-troponin I antibody CDR sequences described herein(see Tables 1 and 2) establish a novel family of troponin I bindingproteins, isolated in accordance with this invention, and comprisingpolypeptides that include the CDR sequences listed in Tables 1 and 2above. To generate and to select CDRs of the invention having preferredtroponin I binding activity, standard methods known in the art forgenerating binding proteins of the present invention and assessing thebinding characteristics thereof may be used, including but not limitedto those specifically described herein.

Anti-Troponin I Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies, such as those of the present invention, are well known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, whichare incorporated herein by reference in their entireties. In addition,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger etal., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454which are incorporated herein by reference in their entireties) bysplicing genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity can be used.

In one embodiment, the chimeric antibodies of the invention are producedby replacing the heavy chain constant region of the antibodies describedabove with a human IgG1 constant region. In a specific embodiment, thechimeric antibody of the invention comprises a heavy chain variableregion (V_(H)) comprising the amino acid sequence of SEQ ID NO:25 and alight chain variable region (V_(L)) comprising the amino acid sequenceof SEQ ID NO:28.

Anti-Troponin I CDR Grafted Antibodies

CDR-grafted antibodies of the invention comprise heavy and light chainvariable region sequences from a human antibody wherein one or more ofthe CDR regions of V_(H) and/or V_(L) are replaced with CDR sequences ofthe murine antibodies of the invention. A framework sequence from anyhuman antibody may serve as the template for CDR grafting. However,straight chain replacement onto such a framework often leads to someloss of binding affinity to the antigen. The more homologous a humanantibody is to the original murine antibody, the less likely thepossibility that combining the murine CDRs with the human framework willintroduce distortions in the CDRs that could reduce affinity. Therefore,it is preferable that the human variable framework that is chosen toreplace the murine variable framework apart from the CDRs have at leasta 65% sequence identity with the murine antibody variable regionframework. It is more preferable that the human and murine variableregions apart from the CDRs have at least 70% sequence identify. It iseven more preferable that the human and murine variable regions apartfrom the CDRs have at least 75% sequence identity. It is most preferablethat the human and murine variable regions apart from the CDRs have atleast 80% sequence identity. Methods for producing chimeric antibodiesare known in the art and discussed in detail in Example 2.2. (See alsoEP 239,400; Intern. Appln. Publication No. WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,352).

Humanized Antibodies

Humanized antibodies are antibody molecules from non-human speciesantibody that bind the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Suchimported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332: 323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994);International Appln. Publication No. WO 91/09967, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106;EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862,5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886,5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089,5,225,539; 4,816,567, each entirely incorporated herein by reference,included references cited therein.

Production of Antibodies and Antibody-Producing Cell Lines

As noted above, preferably, antibodies of the present invention exhibita high capacity to bind specifically to troponin I, e.g., as assessed byany one of several in vitro and in vivo assays known in the art (e.g.,see examples below).

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter et al., U.S. Pat. Nos.5,648,260 and 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions, for example, cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement Clq, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment, at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the invention is derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, alabeled binding protein of the invention can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein. Preferably, the invention relates to crystals of wholeanti-troponin I antibodies and fragments thereof as disclosed herein,and formulations and compositions comprising such crystals. In oneembodiment the crystallized binding protein has a greater half-life invivo than the soluble counterpart of the binding protein. In anotherembodiment; the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in International Appln.Publication No. WO 02/072636, incorporated herein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have importanteffect on the effector function of the Fc domain, with minimal effect onantigen binding or half-life of the antibody (R. Jefferis, Biotechnol.Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Co,M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result inincreased affinity for the antigen (Wallick, S. C., et al., Exp. Med.(1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies. Thecreation of glycosylation site mutants that retain the biologicalactivity but have increased or decreased binding activity are anotherobject of the present invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in International Appln. Publication No. WO 03/016466A2,and U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which isincorporated herein by reference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; International Appln. Publication Nos. WO 03/035835 and WO99/5434280, each of which is incorporated herein by reference in itsentirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S Patent ApplicationPublication Nos. 20040018590 and 20020137134 and International Appln.Publication No. WO 05/100584 A2).

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is preferably engineered to have thethree or more antigen binding sites, and is generally not a naturallyoccurring antibody. The term “multispecific binding protein” refers to abinding protein capable of binding two or more related or unrelatedtargets. Dual variable domain (DVD) binding proteins as used herein, arebinding proteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. Such DVDs may bemonospecific, i.e., capable of binding one antigen or multispecific,i.e., capable of binding two or more antigens. DVD binding proteinscomprising two heavy chain DVD polypeptides and two light chain DVDpolypeptides are referred to a DVD Ig. Each half of a DVD Ig comprises aheavy chain DVD polypeptide, and a light chain DVD polypeptide, and twoantigen binding sites. Each binding site comprises a heavy chainvariable domain and a light chain variable domain with a total of 6 CDRsinvolved in antigen binding per antigen binding site. DVD bindingproteins and methods of making DVD binding proteins are disclosed inU.S. patent application Ser. No. 11/507,050 and incorporated herein byreference.

One aspect of the invention pertains to a DVD binding protein comprisingbinding proteins capable of binding to troponin I. Preferably, the DVDbinding protein is capable of binding troponin I and a second target.The present invention also encompasses triple-variable domain (TVD)binding proteins in which the antibody is capable of binding troponin Ias well as two additional targets (i.e., a second and third target).

In addition to the binding proteins, the present invention is alsodirected to an anti-idiotypic (anti-Id) antibody specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the-binding protein (e.g., antibody ofinterest) or a CDR containing region thereof. The immunized animal willrecognize, and respond to the idiotypic determinants of the immunizingantibody and produce an anti-Id antibody. The anti-Id antibody may alsobe used as an “immunogen” to induce an immune response in yet anotheranimal, producing a so-called anti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

Diagnostic Uses of Anti-Troponin I Antibodies

Given their ability to bind to troponin I, the anti-troponin Iantibodies, or portions thereof, of the invention can be used to detecttroponin I (e.g., in a biological sample such as serum, whole blood,CSF, brain tissue or plasma), using a conventional immunoassaycompetitive or non-competitive assay such as, for example, an enzymelinked immunosorbent assay (ELISA), a radioimmunoassay (RIA), animmunometric assay or tissue immunohistochemistry. The inventiontherefore provides a method for detecting troponin I in a biologicalsample comprising contacting a biological sample with an antibody, orantibody portion, of the invention and detecting either the antibody (orantibody portion) bound to troponin I or unbound antibody (or antibodyportion), to thereby detect troponin I in the biological sample. Theantibody is directly or indirectly labeled with a detectable substanceto facilitate detection of the bound or unbound antibody. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

As an alternative to labeling the antibody, troponin I can be assayed inbiological fluids by a competition immunoassay utilizing recombinanttroponin I standards labeled with a detectable substance and anunlabeled anti-troponin I antibody. In this assay, the biologicalsample, the labeled recombinant troponin I standards and theanti-troponin I antibody are combined, and the amount of labeledrecombinant troponin I standard bound to the unlabeled antibody isdetermined. The amount of troponin I in the biological sample isinversely proportional to the amount of labeled recombinant troponin Istandard bound to the anti-troponin I antibody.

In particular, in one embodiment of the present invention, one or moreof the antibodies of the present invention are coated on a solid phase(or are in a liquid phase). The test or biological sample (e.g., serum,plasma, urine, etc.) is then contacted with the solid phase. If troponinI antigens are present in the sample, such antigens bind to theantibodies on the solid phase and are then detected by either a director indirect method. The direct method comprises simply detectingpresence of the complex itself and thus presence of the antigens. Itshould be noted that one may use the full antibody or a fragment thereofin connection with the antibodies coated on the solid phase. (Forpurposes of the present invention, a “fragment” or “portion” of anantibody is defined as a subunit of the antibody which reacts in thesame manner, functionally, as the full antibody with respect to bindingproperties.)

As mentioned above, in the indirect method, a conjugate is added to thebound antigen. The conjugate comprises a second antibody, which binds tothe bound antigen, attached to a signal-generating compound or label.Should the second antibody bind to the bound antigen, thesignal-generating compound generates a measurable signal. Such signalthen indicates presence of the antigen (i.e., troponin I) in the testsample.

Examples of solid phases used in diagnostic immunoassays are porous andnon-porous materials, latex particles, magnetic particles,microparticles (see U.S. Pat. No. 5,705,330), beads, membranes,microtiter wells and plastic tubes. The choice of solid phase materialand method of labeling the antigen or antibody present in the conjugate,if desired, are determined based upon desired assay format performancecharacteristics.

As noted above, the conjugate (or indicator reagent) will comprise anantibody (or perhaps anti-antibody, depending upon the assay), attachedto a signal-generating compound or label. This signal-generatingcompound or “label” is itself detectable or may be reacted with one ormore additional compounds to generate a detectable product. Examples ofsignal-generating compounds include chromogens, radioisotopes (e.g.,125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g.,acridinium), particles (visible or fluorescent), nucleic acids,complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluro-, or lumo-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful.

As noted above, examples of biological fluids which may be tested by theabove immunoassays include plasma, urine, whole blood, dried wholeblood, serum, cerebrospinal fluid, saliva, tears, nasal washes oraqueous extracts of tissues and cells.

Additionally, it should also be noted that the initial capture antibody(for detecting troponin I antigens) used in the immunoassay may becovalently or non-covalently (e.g., ionic, hydrophobic, etc.) attachedto the solid phase. Linking agents for covalent attachment are known inthe art and may be part of the solid phase or derivatized to it prior tocoating.

Further, the assays and kits of the present invention optionally can beadapted or optimized for point of care assay systems, including Abbott'sPoint of Care (i-STAT™) electrochemical immunoassay system.Immunosensors and methods of manufacturing and operating them insingle-use test devices are described, for example in U.S. Pat. No.5,063,081 and published U. S. Patent Application Nos. 20030170881,20040018577, 20050054078, and 20060160164 (incorporated by referenceherein for their teachings regarding same).

Of course, any of the exemplary formats herein and any assay or kitaccording to the invention can be adapted or optimized for use inautomated and semi-automated systems (including those in which there isa solid phase comprising a microparticle), as described, e.g., in U.S.Pat. Nos. 5,089,424 and 5,006,309, and as, e.g., commercially marketedby Abbott Laboratories (Abbott Park, Ill.) including but not limited toAbbott's ARCHITECT®, AxSYM, IMX, PRISM, and Quantum II platforms, aswell as other platforms.

Other assay formats which may be used for purposes of the presentinvention include, for example, a rapid test, a Western blot, as well asthe use of paramagnetic particles in, for example, an ARCHITECT® assay(see Frank Quinn, The Immunoassay Handbook, Second edition, edited byDavid Wild, pages 363-367, 2001, herein incorporated in its entirety byreference). Such formats are known to those of ordinary skill in theart.

It should also be noted that the elements of the assays described aboveare particularly suitable for use in the form of a kit. The kit may alsocomprise one container such as a vial, bottle or strip. These kits mayalso contain vials or containers of other reagents needed for performingthe assay, such as washing, processing and indicator reagents.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody, or antigen-binding portion thereof, of the invention and apharmaceutically acceptable carrier. The pharmaceutical compositionscomprising antibodies of the invention are for use in, but not limitedto, diagnosing, detecting, or monitoring a disorder, in preventing,treating, managing, or ameliorating of a disorder or one or moresymptoms thereof, and/or in research. In a specific embodiment, acomposition comprises one or more antibodies of the invention. Inanother embodiment, the pharmaceutical composition comprises one or moreantibodies of the invention and one or more prophylactic or therapeuticagents other than antibodies of the invention for treating a disorder inwhich troponin I activity is detrimental. Preferably, the prophylacticor therapeutic agents known to be useful for or having been or currentlybeing used in the prevention, treatment, management, or amelioration ofa disorder or one or more symptoms thereof. In accordance with theseembodiments, the composition may further comprise of a carrier, diluentor excipient.

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and International Appln.Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, andWO 99/66903, each of which is incorporated herein by reference theirentireties. In one embodiment, an antibody of the invention, combinationtherapy, or a composition of the invention is administered usingAlkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.). In a specific embodiment, prophylactic or therapeuticagents of the invention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard .etal., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; International Appln. Publication No. WO 99/15154;and International Appln. Publication No. WO 99/20253. Examples ofpolymers used in sustained release formulations include, but are notlimited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In a preferred embodiment, the polymer usedin a sustained release formulation is inert, free of leachableimpurities, stable on storage, sterile, and biodegradable. In yetanother embodiment, a controlled or sustained release system can beplaced in proximity of the prophylactic or therapeutic target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, International Appln. Publication No. WO 91/05548,International Appln. Publication No. WO 96/20698, Ning et al., 1996,“Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Usinga Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song etal., 1995, “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions,” PDA Journal of Pharmaceutical Science & Technology50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for abFGF Antibody for Cardiovascular Application,” Pro. Intl. Symp. Control.Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proc. Intl Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

It should be understood that the antibodies of the invention or antigenbinding portion thereof can be used alone or in combination with one ormore additional agents, e.g., a therapeutic agent (for example, a smallmolecule or biologic), said additional agent being selected by theskilled artisan for its intended purpose. The additional agent also canbe an agent that imparts a beneficial attribute to the therapeuticcomposition e. g., an agent that affects the viscosity of thecomposition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limiting. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included for purposes of illustration only and are not intended tolimit the scope of the present invention.

EXAMPLES Example I Generation and Isolation of 19C7

Identification of Immunoglobulin Genes

Messenger RNA was isolated from subcloned anti-TnI 19C7-144 hybridomacells. (Hybridoma cell line TnI 19C7 is described in U.S. PatentApplication Publication No. US2006/0018897.) TnI 19C7 mRNA was utilizedin a reverse transcriptase-polymerase chain reaction using a mouse Igprimer set kit purchased from Novagen (Novagen (which is an Affiliate ofMerck KGaA, Darmstadt, Germany), Cat No. 69831-3) with immunoglobulingene specific primers contained in the kit. The resulting PCR productswere sequenced and thus the immunoglobulin variable heavy and variablelight chain genes were identified (see FIG. 2).

Cloning TnI 19C7 Variable Region Genes into pYD41 Vector

A yeast display system was used to express unmutated or wild-typeanti-TnI proteins (described herein infra) and a library of anti-TnIproteins on the yeast surface as a fusion to the yeast protein AGA2. Ayeast display vector called pYD (Invitrogen, Carlsbad, Calif.) was usedas it allows for cloning of the anti-TnI gene at the C-terminus of theAGA2 gene, a yeast mating factor (See, Boder and Wittrup, NatureBiotechnology, 15:553-557 (June 1997)). Other critical features of thepYD vector include a galactose inducible promoter and an epitope tag,V5, on the C-terminus of the inserted anti-TnI gene (see FIG. 12).

The yeast display platform utilizes an antibody format known as thesingle-chain variable fragment. In the scFv format, the variable heavydomain is connected to the variable light domain through a flexiblelinker (variable heavy domain—Linker GPAKELTPLKEAKVS (SEQ IDNO:58)—variable light domain).

PCR single overlap extension (SOE) was used to combine the variableheavy (VH) and the variable light genes (VL) for the TnI 19C7 scFvconstruct (FIG. 2, and SEQ ID NOs:54 and 55). The TnI 19C7 scFv DNA wascloned into the yeast display vector pYD41 using vector restrictionsites Sfil and Xhol. The pYD41-TnI 19C7scFv vector was transformed intoDHSα E. coli. Plasmid DNA was then isolated from the E. coli and the TnI19C7 scFv insert was sequenced to ensure the scFv was cloned in framewith the AGA2 protein.

The cloning site for the scFv into the yeast display vector pYD41 is inan ORF that includes the following genes: AGA2-tether linker 41-X pressepitope tag-TnI 19C7 variable heavy chain-Linker 40-TnI 19C7 variablelight chain-V5 epitope tag—Six His tag (SEQ ID NO: 29). In addition, theyeast strain EBY100 is a tryptophan auxotroph and the pYD41 vectorencodes tryptophan as the system's selectable marker.

Transformation into Saccharomyces cerevisiae Strain EBY100

Yeast display plasmid, pYD41-TnI 19C7 scFv, was transformed into S.cerevisiae EBY100 using the Gietz and Schiestl Method (See Schiestl andGietz, Current Genetics, 16(5-6):339-46 (Dec. 1989)). Dilutions of thetransformation reaction were plated on selective glucose plates (2%glucose (0.67% yeast nitrogen base, 0.105% HSM-trp -ura, 1.8% bacterialagar, 18.2% sorbitol, 0.86% NaH₂PO₄ H₂O, 1.02% Na₂HPO₄ 7H₂O)) andincubated at 30° C. for 48-72 hours. Selective glucose media wasinoculated with individual colonies and grown shaking at 30° C. for16-20 hours. Protein expression was induced in colonies by transferring0.5 OD600 of cells/ml (le7cells/0.50 D/ml) to selective galactose media.Colonies were shaken at 20° C. for 16-24 hours and then analyzed by theFACS Aria flow cytometer for binding to scTnI-C-2 and anti-V5. (Itshould be noted that scTnI-C-2 is a linked, single-cahin TnI(28-110aa)-linker-TnC (1-160aa) from Spectral Diagnostics, Toronto,Canada. ScTnI-C-2 is abbreviated as “scTnI-C” for purposes of thepresent discussion.) For flow cytometry assays, yeast cells expressingTnI 19C7 scFv incubated with scTnI-C-2 or anti-V5 followed by eitheranti-troponin mAb and goat anti mouse-phycoerythrin (GAM:PE) (FIG. 3B)or GAM:PE respectively (FIG. 3A). The flow cytometry histogramsillustrate the full-length expression of TnI 19C7 scFv as detected byanti-V5 and the ability of TnI 19C7 scFv to bind to scTnI-C-2.

Off-Rate Analysis for TnI 19C7 scFv and TnI 19C7 Variants on Yeast

Off-rate measurements of TnI 19C7 scFv and TnI 19C7 variants on yeastwere measured by incubating 0.050 D yeast (1×10⁶ cells) with 50 nMscTnI-C-2 for 30-60 minutes at room temperature. Cells were then washedtwice with blocking buffer containing phosphate buffered saline pH 6.8with 2% bovine serum albumin and 0.2% Standapol ES-1 (PBS/BSA/Standapol)and incubated at room temperature for varying amounts of time (0, 0.25hr, 0.5 hr, 1 hr, 2 hr, 4.25 hr, 25.5 hr, 50 hr 75 hr and 144 hr (seeFIG. 4). At each individual time point, yeast cells were transferred toice to halt the reaction. Cells were then washed twice with blockingbuffer and suspended in the next staining reagent, specifically,anti-TnI mAb 8E10 at 100 nM. Cells were incubated on ice for 30 minutes,washed twice and then incubated with goat anti mouse-phycoerythrin(GAM:PE). Finally, the cells were washed and analyzed on the FACS Ariaflow cytometer. FIG. 4 shows the off-rate data plotted as meanfluorescence units (“MFU”) versus time (in seconds). A first order decayequation was used to fit the data. The off-rate, m2 in the equationshown in FIG. 4, was fitted to 0.007 sec⁻¹. The TnI 19C7 scFv half-life(t_(1/2)) was approximately 8.5 min (t_(1/2)=1 n2/k_(off)).

An off-rate sorting strategy was used to identify off-rate improved TnI19C7 variants from mutagenic libraries. Therefore, the TnI 19C7 scFv,unmutated or wildtype (“wt”), half-life was used to determine theappropriate time to sort the mutagenic libraries. TnI 19C7 mutageniclibraries were sorted approximately 9 min after washing cells free ofscTnI-C-2 with the same assay conditions described for wt TnI 19C7 scFv.

Equilibrium Disassociation (KD) Analysis for TnI 19C7 scFv and TnI

KD measurements of TnI 19C7 scFv and TnI 19C7 variants on yeast weremeasured by incubating 0.05 OD yeast (1×10⁶ cells) with varyingconcentrations of scTnI-C-2 for 45-60 minutes at room temperature.Blocking buffer containing phosphate buffered saline pH 6.8 with 2%bovine serum albumin and 0.2% Standapol ES-1 (PBS/BSA/Standapol) wasused for washes and reagent dilutions. Cells were then washed twice andincubated for 30 min with anti-TnI mAb, 8E10. Cells were washed againand incubated with goat anti-mouse phycoerythrin for 30 min. Finally,cells were washed and analyzed on the FACS Aria flow cytometer (see FIG.5). FIG. 5 shows the KD data plotted as normalized mean fluorescenceunits (“MFU”) versus concentration scTn-I-C-2 (in Molarity). Theantibody-normalized, antigen-binding mean fluorescence intensity wasplotted against antigen concentration and a non-linear least squares fit(y=m1+m2*m0/(m3+m0)) was used to determine K_(D).

Generation of TnI 19C7 Spiked CDR Libraries

Mutagenesis was directed to the three heavy and three light chaincomplementary determining regions (CDR) of antibody TnI 19C7 since theseloops are the major antigen contact sites. CDR loop lengths andnumbering were defined using Kabat and Oxford's Molecular AbM modelingnomenclature. Individual libraries were composed such that randommutations are incorporated at each amino acid position of the CDR for asingle library. A total of six libraries were generated corresponding toone library per each CDR.

Libraries were generated by combining Sfil/Xhol digested pYD41 vectorand PCR products with chemically competent EBY100 yeast (see FIG. 7).Two PCR products were generated for each CDR library to allow for PCRsorting and homologous recombination into yeast. One PCR product used aprimer that was designed, such that for the entire length of the CDR, a70% wild type to 30% other nucleotide ratio was used in the primersynthesis. This product was called the spiked (sp) product (see FIG. 7).The second PCR product was designed to include the remaining portion ofthe scFv gene. The two PCR products were combined and used to generate asingle-chain variable fragment or a scFv product. Digested vector (1 ug)and the scFv PCR products (5 ug) were combined with EBY100 yeast(5.2e8-6.4e8 cells) and transformed using electroporation. The scFv PCRproduct and the pYD41 digested vector cyclize during transformation dueto homologous recombination facilitated by the nucleotide overlap andthe mechanism of yeast endogenous gap repair. Libraries were grown at30° C. for 48-72 hours in selective glucose media and passed again inselective glucose media prior to induction of protein expression forlibrary sorting.

TnI 19C7 Mutagenic CDR Libraries

TnI 19C7 libraries were sorted based on an off-rate sorting strategy.TnI 19C7 CDR mutagenic libraries were induced in galactose expressionmedia at 20° C. for 18-24 hours. TnI 19C7 scFv and TnI 19C7 libraries onyeast were incubated with 25-50 nM scTnI-C-2 for 10-15 minutes at roomtemperature. Cells were then washed twice with blocking buffercontaining phosphate buffered saline pH 6.8 with 2% bovine serum albuminand 0.2% Standapol ES-1 (PBS/BSA/Standapol) and incubated at roomtemperature for 8 min. Yeast cells were transferred to ice to halt thereaction. Cells were then washed twice with blocking buffer andsuspended in the next staining reagent, specifically, anti-TnI mAb 8E10at 100 nM and anti-V5 at 1.5-2 ug/ml. Cells were incubated on ice for 30minutes, washed twice and then incubated with 1:200 goat antimouseIgG2a-phycoerythrin (GAMIgG2a:PE) and with 1:200 dilution goat antimouseIgG1-Alexa Fluor488 (GAMIgG1:488). Finally, the cells were washed,analyzed, and sorted on the FACS Aria flow cytometer. Sort gates wereset based on unmutated TnI 19C7 binding at 8-9 min with a gate set tosort full-length TnI binding clones. Each sort collected the top0.1-0.5% of the TnI binding population. Sorted cells were grown inselective glucose media and grown 18-24 hours at 30° C. Sort 1 cellswere induced and sorting was repeated for two or three additionalrounds.

After the last sort, sorted cells were plated onto selective glucoseplates and placed at 30° C. for 72 hours. Individual yeast colonies fromthese libraries were inoculated in selective glucose media,cryopreserved and induced in selective galactose media. Individualcolonies were then characterized and ranked in an off-rate assay. TnI19C7 AM4 was isolated and identified from this sorting output.

Generation and Analysis of TnI 19C7 Combinatorial Mutant Clones

Clones that were characterized for off-rate from each master CDR libraryor the total master CDR library output were used to construct scFv genescontaining different pairings of the individual mutations. This approachenabled determination of whether the binding properties were furtherenhanced upon combining individual mutations. Combinatorial clonescontaining various mutations in each CDR region were constructed by PCRamplification and combined using routine techniques known to thoseskilled in the art. Combinatorial mutant libraries were transformed intoyeast as described above and sorted two times using off-rate and KDselection pressures. For KD selection, 100 pM (round 1) and 50 pM (round2) scTnI-C were used in the KD experiment as described above (FIG. 5).For off-rate sorting, sorting was conducted as described above withincubation times following washing away of antigen of 3 hr 40 min(round 1) and 4 hrs 25 min (round 2). Sort gates were set based onunmutated TnI 19C7 binding for each condition with a gate set to sortfull-length TnI binding clones. Each sort collected the top 0.1% of theTnI binding population. Sorted cells were grown in selective glucosemedia for 18-24 hours at 30° C. Sort 1 cells were induced and sortingwas repeated for one additional round.

After the last sort, sorted cells were plated onto selective glucoseplates and placed at 30° C. for 72 hours. Individual yeast colonies fromthese libraries were inoculated in selective glucose media,cryopreserved and induced in selective galactose media. Individualcolonies were then characterized and ranked in an off-rate assay. TnI19C7 AM1, AM2, and AM3 were isolated and identified from thiscombinatorial library,

Analysis of Selected TnI 19C7 Variants

Selected clones were initially characterized for improvements in KD asdescribed above for wild type TnI 19C7 scFv. FIG. 9 shows the scFv KDvalues determined for four selected clones. The TnI 19C7 AM1 cloneexhibited the most improved binding at 0.36 nM compared to thewild-typeTnI 19C7 antibody 1.7 nM.

Selected TnI 19C7 scFv variants were sequenced to determine the aminoacid mutations being expressed. Initially, plasmid DNA was isolated fromyeast suspension cultures using a yeast mini-prep kit (Cat No. D2001,Zymo Research Orange, Calif.). In order to obtain sequencing gradeplasmid DNA, plasmid from the yeast mini-prep kit was transformed intoDH5αE. coli, and then purified from culture using E. coli mini-prep kits(Qiagen). Pure plasmid DNA was then sequenced using pYD41 vectorspecific primers (pYD41 for -TAGCATGACTGGTGGACAGC (SEQ ID NO:59) andpYD41 rev-CGTAGAATCGAGACCGAG (SEQ ID NO:60)). Amino acid sequence datafor TnI 19C7 scFv variants is shown in FIG. 13. Position numbers refersto amino acid position in the respective CDR (as numbered using Kabatmethod).

Overall the source of sequence diversity from the wild-type clone wasfound in the CDR L2 and CDR H1, whereas CDR L1 and H3 folded into aconsensus motif. The CDR L3 and CDR H2 remained unmutated. The sequencedata for CDR H1 indicated a preference for a conservative change atposition 34 from isoleucine to leucine as identified in the 3 clonesisolated from the combinatorial library. The consensus sequence for CDRH3 indicated a strong preference at position 100a for tyrosine insteadof tryptophan, at position 101 for threonine instead of alanine, and atposition 102 for aspartate instead of tyrosine as identified in the 3clones isolated from the combinatorial library. From the master CDRsorting in which clone 19C7 AM4 was identified, the CDR H3 was the onlyCDR with mutations that were different than the combinatorial consensusset of sequences. Specifically the mutations were Ala96Phe, Tyr99Ser,Trp100aAla, and Tyr102Asp.

The consensus sequence data for CDR L1 indicated a preference forthreonine at position 25, lysine at position 27, asparagine at position28, valine at position 29, and histidine at position 34. For the CDR L2,each clone has unique or no mutations with only TnI 19C7 AM1 and AM2sharing only the Ser54Arg mutation.

Cloning and Soluble Expression of TnI 19C7 Chimeric Antibodies in aTransient or Stable Expression System

Selected TnI 19C7 variants were converted to chimeric mouse-mouseIgG_(2a)/mouse kappa and/or mouse-human IgG₁/human kappa antibodiesthrough cloning of the TnI 19C7 variable domains into the transientexpression vector system called pBOS (Abbott Bioresearch Center,Worcester, Mass.). More specifically, PCR was used to amplify thevariable heavy and variable light chain genes with restriction sites forcloning into separate pBOS vectors (Mizushima and Nagata, Nucleic AcidsResearch, 18:5322 (1990)). The variable heavy and variable light geneswere ligated in digested and dephosphorylated vector and transformedinto DH5α E. coli. Plasmid DNA was purified from E. coli and transfectedinto 293H cells using PEI (1 mg/ml). Transient antibody was expressedfor the following TnI 19C7 variants: TnI 19C7 wt/AM1, AM2, AM3 and AM4.

For example, using the pBOS-TnI 19C7 AM1 heavy and light vectors, astable CHO cell line plasmid was created in a two-step cloningprocedure. First, variable heavy chain and variable light genes wereligated in frame to the human constant genes in pBV and pJV plasmids(Abbott Bioresearch Center, Worcester; MA), respectively, using therestriction enzymes SrfI/NotI. Ligation reactions were transformed intoDH5αE. coli and plasmid DNA was subsequently isolated from individualcolonies. The pBV-TnI 19C7 mouse variable heavy-human IgG1 and pJV-TnI19C7 mouse variable light-human kappa were sequenced at the cloningsites.

The second cloning step involved combining the heavy chain IgG₁ genesand the light chain kappa genes into a single stable cell line vector.The pBV-TnI19C7 AM1 human IgG1 and•pJV-TnI 19C7 AM1 human kappa vectorswere digested with AscI/PacI. The VL-human kappa constant and theVH-human IgG1 constant DNA fragments were gel purified and ligated toproduce the stable cell line vector called pBJ-TnI19C7 AM1. The pBJ-TnI19C7 AM1 human heavy/light chimeric plasmid was transformed into CHOcells using a lipofectamine (Invitrogen) protocol. Stable cell lineswere subcloned from initial transformation. A stable CHO cell line hasbeen developed for the clone AM1 (also referred to as “TnI 19C7AM1hG1kCHO204”) and was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 on Feb.11, 2009 and received deposit designation PTA-9816.

Example II

Relative Affinity of Troponin I Clone 19C7 Wild Type and AffinityMatured Antibodies

Troponin I clone 19C7 wild type (full mouse construct) and affinitymatured (human constant region) antibodies were evaluated for relativeaffinity in a microtiter enzyme immunoassay. 96-well assay plates (NUNCCorporation, Rochester, N.Y.) were coated by adding 100 uL/well of a 2ug/mL solution of either sheep anti-mouse IgG Fcγ specific antibody(Jackson ImmunoResearch, West Grove, Pa.) or donkey anti-human IgG Fcγfragment specific antibody (Jackson ImmunoResearch). Both antibodieswere diluted in phosphate buffered saline (PBS, Abbott Laboratories,Abbott Park, Ill.). The assay plates were incubated overnight at 15-30deg C. The next day the coating reagent was removed and 200 uL/well ofBSA solution (bovine serum albumin [Abbott Laboratories] diluted in PBS)was added. The BSA solution was incubated in the assay wells for 30minutes at 15-30 deg C, removed and the assay wells washed by adding 300uL/well distilled water (dH2O, Abbott Laboratories) and aspirating forthree wash cycles. Next, 100 uL/well test samples were added. Testsamples were prepared by creating an initial 2 ug/mL solution (in BSAsolution) of each antibody, followed by log 3 dilutions, in BSAsolution. The test samples were incubated for 2-3 hours at 15-30 deg C.after which they were aspirated away and the wells washed with dH2O asdescribed above. Next, 100 uL/well of test antigen solutions were addedto each assay well. The test antigen solutions were created by firstpreparing a 1000 ng/mL solution of scTnI-C-2 (aa 28-100 of cardiactroponin I linked to full length cardiac troponin C, SpectralDiagnostics) in BSA solution, followed by log 2 dilutions. The antigensolutions were incubated in the assay wells for 10 minutes at 15-30 degC and then removed by slapping out the solutions. The assay plates werethen washed with dH2O as previously to described. Next, 100 uL/well ofbiotin labeled goat anti-troponin I antibody (HyTest, diluted to 500ng/mL in BSA solution) was added to each assay well and incubated for 30minutes at 15-30 deg C. The antibody was then aspirated away and thewells washed with dH2O as described. Next, 100 uL/well of a 200 ng/mLsolution (in BSA solution) of horse radish peroxidase labeledstreptavidin (SA-HRPO, Jackson ImmunoResearch) was added and incubatedfor 30 minutes at 15-deg C. The SA-HRPO reagent was then, aspirated awayand the plates washed as described. Next, substrate solution wasprepared by dissolving 1 OPD tablet per 10 mL OPD diluent(o-phenylenediamine, both Abbott Laboratories). 100 uL/well of theprepared substrate solution was added to the assay plates, incubated forabout 4-5 minutes and then the reaction quenched by adding 100 uL/well1N sulfuric acid (Abbott Laboratories). The resulting signal was read at492 nm using an optical fluorometer. Results from the experiment wereplotted using kaleidagraph software. The Ag₅₀) value (the concentrationof antigen at 50% of maximal binding) was determined and used to comparethe antibodies for relative affinity to the tested antigen.

Example III

Use of Monoclonal Antibody 19C7 in an Immunoassay

Troponin I clone 19C7 wild type (full mouse construct) and affinitymatured (human constant region) antibodies were evaluated for relativeaffinity on the ARCHITECT® immunoassay analyzer (Abbott Laboratories).The assay was fully automated, and the analyzer performs all steps.Magnetic microparticles coated with a mouse anti-troponin I antibody(Abbott Laboratories, Abbott Park, Ill.) were mixed with varying levelsof scTnI-C-2 (aa 28-100 of cardiac troponin I linked to full lengthcardiac troponin C (Spectral Diagnostics) and incubated for 18 minutesat 15-30 deg C. During this time, the microparticle coated antibodybound the scTnI-C-2. The microparticles were then attracted to a magnet,the remaining assay solution was aspirated, and the particles washedwith assay diluent (Abbott Laboratories, Abbott Park, Ill.). Next, thewild type or affinity matured 19C7 antibodies, all of which were labeledwith acridinium (Abbott Laboratories, Abbott Park, Ill.) were added tothe microparticles and incubated for 4 minutes at 15-30 deg C. Next, themicroparticles were attracted to a magnet, the remaining assay solutionwas aspirated, and the particles were washed with assay diluent. Signal(relative light units) was generated by the addition of pre-trigger andtrigger solutions (both Abbott Laboratories). Signal ratios werecalculated and used to compare antibodies. As can be established basedupon the results shown in FIG. 11, AM1 antibody gave a better signalthan wild-type antibody.

1-35. (canceled)
 36. A composition comprising a detectable label boundto a Troponin I binding antibody, wherein said antibody comprises a CDR1sequence of SEQ ID NO:52, a CDR2 sequence of SEQ ID NO: 53, and a CDR3sequence of SEQ ID NO:54, and a light chain that comprises a CDR1sequence of SEQ ID NO:55, a CDR2 sequence of SEQ ID NO: 56, and a CDR3sequence of SEQ ID NO:57.
 37. A composition comprising a detectablelabel bound a Troponin I binding antibody, wherein said Troponin Ibinding antibody is a variant of TnI 19C7 and wherein variant comprisesan antigen binding domain in which there is a mutation in the heavychain CDR1 and CDR3 and a mutation in the light chain CDR1 and CDR2 ascompared to the sequences of those respective CDRs in TnI 19C7 whichconsist of SEQ ID NO: 30 and SEQ ID NO:35 as CDR1 and CDR3 of TnI 19C7heavy chain and SEQ ID NO: 40 and SEQ ID NO:45 as CDR1 and CDR2 of TnI19C7 light chain, and wherein said antibody variant binds to Troponin I.38. The composition of claim 36, wherein said variant is a humanizedantibody comprising a human acceptor framework.
 39. The composition ofclaim 36, wherein the amino acid sequence of the variable heavy chain ofsaid antigen binding domain has at least 98% identity to SEQ ID NO:25.40. The composition of claim 36, wherein the amino acid sequence of thevariable heavy chain of said antigen binding domain has at least 99%identity to SEQ ID NO:25.
 41. The composition of claim 36, wherein theamino acid sequence of the variable heavy chain of said antigen bindingdomain has a sequence of SEQ ID NO:25.
 42. The composition of claim 36,wherein the amino acid sequence of the variable light chain of saidantigen binding domain has at least 94% identity to SEQ ID NO:28. 43.The composition of claim 36, wherein the amino acid sequence of thevariable light chain of said antigen binding domain has at least 95%identity to SEQ ID NO:28.
 44. The composition of claim 36, wherein theamino acid sequence of the variable light chain of said antigen bindingdomain has at least 96% identity to SEQ ID NO:28.
 45. The composition ofclaim 36, wherein the amino acid sequence of the variable light chain ofsaid antigen binding domain has at least 97% identity to SEQ ID NO:28.46. The composition of claim 36, wherein the amino acid sequence of thevariable light chain of said antigen binding domain has at least 98%identity to SEQ ID NO:28.
 47. The composition of claim 36, wherein theamino acid sequence of the variable light chain of said antigen bindingdomain has at least 99% identity to SEQ ID NO:28.
 48. The composition ofclaim 36, wherein the amino acid sequence of the variable light chain ofsaid antigen binding domain has a sequence of SEQ ID NO:28.
 49. Thecomposition of claim 36, wherein said antibody variant comprises a heavychain sequence has at least 98% identity to SEQ ID NO:25 and a lightchain sequence that has at least 94% identity to SEQ ID NO:28.
 50. Thecomposition of claim 36, wherein said detectable label is asignal-generating compound capable of generating a detectable signalselected from the group consisting of an enzyme, a prosthetic groupcomplex, a fluorescent material, a luminescent material and aradioactive material.
 51. The composition of claim 50, wherein saidenzymes are selected from the group consisting of horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase.
 52. The composition of claim 50, wherein saidprosthetic group complex is selected from the group consisting ofstreptavidin/biotin and avidin/biotin.
 53. The composition of claim 50,wherein said fluorescent material is selected from the group consistingof umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.54. The composition of claim 50, wherein said luminescent material isluminol.
 55. The composition of claim 50, wherein said radioactivematerial is selected from the group consisting of 3H, 14C, 35S, 90Y,99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.
 56. The composition ofclaim 50, wherein said detectable label is acridinium.
 57. A compositioncomprising a solid support coated with a Troponin I binding antibody,wherein said antibody comprises a CDR1 sequence of SEQ ID NO:52, a CDR2sequence of SEQ ID NO: 53, and a CDR3 sequence of SEQ ID NO:54, and alight chain that comprises a CDR1 sequence of SEQ ID NO:55, a CDR2sequence of SEQ ID NO: 56, and a CDR3 sequence of SEQ ID NO:57.
 58. Acomposition comprising a solid support coated with a Troponin I bindingantibody, wherein said Troponin I binding antibody is a variant of TnI19C7 and wherein said variant comprises a mutation of at least one ofheavy chain CDR1 of TnI 19C7 comprising SEQ ID NO: 30, heavy chain CDR3of TnI 19C7 comprising SEQ ID NO:35, light chain CDR1 of TnI 19C7comprising SEQ ID NO: 40, and light chain CDR2 of TnI 19C7 comprising ofSEQ ID NO:45, and wherein said antibody variant binds to troponin I. 59.The composition of claim 58, wherein said variant comprises a mutationin the heavy chain CDR1 and CDR3 and a mutation in the light chain CDR1and CDR2 as compared to the sequences of those respective CDRs in TnI19C7 which consist of SEQ ID NO: 30 and SEQ ID NO:35 as CDR1 and CDR3 ofTnI 19C7 heavy chain and SEQ ID NO: 40 and SEQ ID NO:45 as CDR1 and CDR2of TnI 19C7 light chain, and wherein said antibody variant binds totroponin I.
 60. The composition of claim 57, wherein said antibodyvariant comprises an antigen binding domain which comprises a heavychain CDR1 sequence of SEQ ID NO: 52, a heavy chain CDR3 sequence of SEQID NO: 54 and a light chain CDR1 sequence of SEQ ID NO: 55 and a lightchain CDR2 sequence of SEQ ID NO:56.
 61. The composition of claim 57,wherein said variant is a humanized antibody comprising a human acceptorframework.
 62. The composition of claim 57, wherein the amino acidsequence of the variable heavy chain of said antigen binding domain hasat least 98% identity to SEQ ID NO:25.
 63. The composition of claim 57,wherein the amino acid sequence of the variable heavy chain of saidantigen binding domain has at least 99% identity to SEQ ID NO:25. 64.The composition of claim 57, wherein the amino acid sequence of thevariable heavy chain of said antigen binding domain has a sequence ofSEQ ID NO:25.
 65. The composition of claim 57, wherein the amino acidsequence of the variable light chain of said antigen binding domain hasat least 94% identity to SEQ ID NO:28.
 66. The composition of claim 57,wherein the amino acid sequence of the variable light chain of saidantigen binding domain has at least 95% identity to SEQ ID NO:28. 67.The composition of claim 57, wherein the amino acid sequence of thevariable light chain of said antigen binding domain has at least 96%identity to SEQ ID NO:28.
 68. The composition of claim 57, wherein theamino acid sequence of the variable light chain of said antigen bindingdomain has at least 97% identity to SEQ ID NO:28.
 69. The composition ofclaim 57, wherein the amino acid sequence of the variable light chain ofsaid antigen binding domain has at least 98% identity to SEQ ID NO:28.70. The composition of claim 57, wherein the amino acid sequence of thevariable light chain of said antigen binding domain has at least 99%identity to SEQ ID NO:28.
 71. The composition of claim 57, wherein theamino acid sequence of the variable light chain of said antigen bindingdomain has a sequence of SEQ ID NO:28.
 72. The composition of claim 57,wherein said antibody variant comprises a heavy chain sequence has atleast 98% identity to SEQ ID NO:25 and a light chain sequence that hasat least 94% identity to SEQ ID NO:28.
 73. The composition of claim 57,wherein said solid support is selected from the group consisting oflatex particles, magnetic particles, microparticles, beads, membranes,microtiter wells and plastic tubes.
 74. The composition of claim 73,wherein said solid support is magnetic.
 75. The composition of claim 73,wherein said solid support is a microparticle.
 76. A method of preparinga reagent for use in an immunoassay for detection of troponin I in asample, comprising detectably labeling a Troponin I binding antibody,wherein said antibody comprises a CDR1 sequence of SEQ ID NO:52, a CDR2sequence of SEQ ID NO: 53, and a CDR3 sequence of SEQ ID NO:54, and alight chain that comprises a CDR1 sequence of SEQ ID NO:55, a CDR2sequence of SEQ ID NO: 56, and a CDR3 sequence of SEQ ID NO:57.
 77. Amethod of preparing a reagent for use in an immunoassay for detection oftroponin I in a sample, comprising detectably labeling a Troponin Ibinding antibody, wherein said Troponin I binding antibody is a variantof TnI 19C7 and wherein variant comprises an antigen binding domain inwhich there is a mutation in the heavy chain CDR1 and CDR3 and amutation in the light chain CDR1 and CDR2 as compared to the sequencesof those respective CDRs in TnI 19C7 which consist of SEQ ID NO: 30 andSEQ ID NO:35 as CDR1 and CDR3 of TnI 19C7 heavy chain and SEQ ID NO: 40and SEQ ID NO:45 as CDR1 and CDR2 of TnI 19C7 light chain, and whereinsaid antibody variant binds to troponin I.
 78. A method of preparing areagent for use in an immunoassay for detection of troponin I in asample, comprising conjugating to a solid support a Troponin I bindingantibody, wherein said antibody comprises a CDR1 sequence of SEQ IDNO:52, a CDR2 sequence of SEQ ID NO: 53, and a CDR3 sequence of SEQ IDNO:54, and a light chain that comprises a CDR1 sequence of SEQ ID NO:55,a CDR2 sequence of SEQ ID NO: 56, and a CDR3 sequence of SEQ ID NO:57.79. A method of preparing a reagent for use in an immunoassay fordetection of troponin I in a sample, comprising conjugating to a solidsupport a Troponin I binding antibody, wherein said Troponin I bindingantibody is a variant of TnI 19C7 and wherein variant comprises anantigen binding domain in which there is a mutation in the heavy chainCDR1 and CDR3 and a mutation in the light chain CDR1 and CDR2 ascompared to the sequences of those respective CDRs in TnI 19C7 whichconsist of SEQ ID NO: 30 and SEQ ID NO:35 as CDR1 and CDR3 of TnI 19C7heavy chain and SEQ ID NO: 40 and SEQ ID NO:45 as CDR1 and CDR2 of TnI19C7 light chain, and wherein said antibody variant binds to troponin I.80. A binding protein produced by culturing a host cell referred to asTnI 19C7 AM1 hG1 CHO 204, designated by American Type Culture Collection(ATCC) deposit Number PTA-9816 under conditions sufficient to producesaid binding protein.